【作者】 孔静；

【导师】 吴硕东；

【作者基本信息】 中国医科大学 ， 外科学， 2009， 博士

【摘要】 胆色素结石形成机制一直是国内外胆道疾病领域的研究热点,有研究表明胆汁成分的改变和胆道感染在胆色素结石形成中发挥了核心作用。以往的研究多是以感染胆汁为中心,通过检测胆汁、胆石成分,围绕Maki学说进行讨论,忽略了感染状态下可能导致肝细胞原发性分泌及原胆汁成分的改变。国内学者近些年来对胆色素结石的发病机理展开了广泛的研究,内容涉及免疫、微量元素、氧自由基、氨基酸含量、游离脂肪酸含量、金属元素在内的多方面研究,但尚无突破性进展。近半个世纪的研究证明,胆色素结石形成必须有三个必要条件和一个中心环节。必要条件是胆汁中游离胆色素增多、胆汁中钙离子增加及助溶因素减弱。中心环节是胆色素钙的沉淀与溶解平衡失调。胆汁的主要成分是胆固醇、胆汁酸和磷脂,它们都是经肝细胞胆小管侧膜分泌入胆汁。作为胆色素结石的关键成分胆色素也是通过肝细胞胆小管侧膜转运蛋白分泌入胆汁。目前关于肝细胞的原发性分泌状态改变导致胆汁成分改变在胆色素结石形成过程中作用的研究甚少。我们通过建立豚鼠胆色素结石模型,观察胆色素结石形成过程中肝细胞胆小管侧膜转运蛋白的表达改变,同时观察胆色素结石形成过程中胆汁主要成分的变化情况,进一步从肝脏原发性分泌状态探索胆色素结石形成的机制。另外采用液相色谱-质谱联用技术做胆汁代谢组学检测,将成石豚鼠胆汁与未成石豚鼠胆汁的代谢图谱进行对比研究分析,成石胆汁与未成石胆汁内代谢物得到分离,并将对二组分离做出贡献的代谢物进一步分析,初步鉴定在胆色素结石豚鼠胆汁中产生了较大差异的代谢物,推测其可能的分子式及结构,并探讨它们在胆色素结石形成过程中的作用。一、实验对象健康豚鼠45只,雌性23只,雄性22只,体质量210～250g,随机分为2组:(对照组/CON组15只,致石组/PS组30只),适应性饲养一周后,分别给予普通饲料及致石饲料。致石周期为8周,饲养期结束后观察各指标。二、标本采集10%水合氯醛腹腔注射麻醉后采集豚鼠肝脏标本、胆囊胆汁标本、胆石标本,-80℃保存,另取部分肝脏标本保存在甲醛溶液中。三、检测指标1.致石率评定及红外光谱结石定性:解剖显露豚鼠胆囊,肉眼即可清晰辨别胆囊结石形成情况,采用红外光谱溴化钾压片法,对结石成分进行定性,统计结石形成率。2.免疫组化方法研究肝细胞胆小管侧膜胆汁酸转运蛋白(Abcbll):肝脏组织制作石蜡切片,免疫组化方法观察肝细胞胆小管侧膜Abcbll分布情况。3.免疫组化方法研究肝细胞胆小管侧膜二价胆盐及结合胆色素转运蛋白(Abcc2):方法同上。4.免疫组化方法研究肝细胞胆小管侧膜磷脂转运蛋白(Abcb4):方法同上。5.Western Blot法研究肝细胞胆小管侧膜胆汁酸转运蛋白Abcb11转录后的改变:取肝脏组织标本,提取膜蛋白,SDS凝胶电泳,ECL化学发光法检测肝细胞胆小管侧膜胆汁酸转运蛋白在转录后水平的变化情况。6.Western Blot法研究肝细胞胆小管侧膜二价胆盐及结合胆色素转运蛋白Abcc2转录后的改变:方法同上。7.Western Blot法研究肝细胞胆小管侧膜磷脂转运蛋白Abcb4转录后的改变:方法同上。8.RT-PCR法研究肝细胞胆小管侧膜胆汁酸转运蛋白Abcb11在mRNA水平上的改变:取肝组织标本,提取肝脏组织总RNA,扩增目的基因,观察肝细胞胆小管侧膜胆汁酸转运蛋白在mRNA水平的变化情况。9.RT-PCR法研究肝细胞胆小管侧膜二价胆盐及结合胆色素转运蛋白Abcc2在mRNA水平上的改变:方法同上。10.RT-PCR法研究肝脏胆小管侧膜胆固醇转运蛋白Abcg5及Abcg8在mRNA水平上的改变:方法同上。11.胆汁生化学检测。12.液质联用胆汁代谢组学研究:胆汁应用高效液相色谱分离后,行质谱分析。实验结果一、豚鼠胆囊胆色素结石模型构建情况对照组豚鼠成石率为14.29%,致石组豚鼠成石率为68.24%,明显高于对照组。结石含有胆色素和胆固醇成分,并以胆色素成分为主。二、豚鼠肝细胞胆小管侧膜转运蛋白的表达变化1、免疫组化结果显示豚鼠肝细胞胆小管侧膜上有棕色颗粒分布,提示肝细胞胆小管侧膜有相应蛋白表达。与对照组豚鼠相比,致石组肝细胞Abcb11(t=5.589,P＜0.01)、Abcc2(t=5.468,P＜0.01)表达下降,着色少;Abcb4表达略有上升,但无统计学意义。致石组中与未成石豚鼠相比,成石豚鼠肝细胞Abcb11(t=8.620,P＜0.01)、Abcc2(t=7.786,P＜0.01)表达下降明显。2、Western Blot结果显示,与对照组相比,致石组豚鼠肝细胞胆小管侧膜转运蛋白Abcb11(t=8.412,P＜0.01)、Abcc2(t=7.894,P＜0.01)表达量下降;致石组中成石豚鼠与未成石豚鼠相比肝细胞胆小管侧膜转运蛋白Abcb11(t=6.042,P＜0.01)、Abcc2(t=9.764,P＜0.01)表达量亦有明显下降;而不同组中豚鼠Abcb4表达无改变。3.采用RT-PCR技术检测肝细胞胆小管侧膜转运蛋白在转录水平上的变化,与对照组相比,致石组豚鼠的肝细胞胆小管侧膜Abcb11(t=6.885,P＜0.01)和Abcc2(t=6.794,P＜0.01)mRNA水平表达都比较低。致石组中成石豚鼠与未成石豚鼠相比肝细胞胆小管侧膜转运蛋白Abcb11(t=2.824,P＜0.05)及Abcc2(t=2.503,P＜0.05)mRNA水平的表达量亦有明显下降,统计学有差异。而各组中胆固醇转运蛋白Abcg5及Abcg8在mRNA水平表达未见有明显差异。三、各组豚鼠胆汁主要成分的变化生化检测结果显示,与对照组相比,致石组豚鼠胆汁中总胆色素、结合胆色素及游离胆色素水平均升高,其中总胆色素(t=3.722,P＜0.01)及游离胆色素(t=3.727,P＜0.01)升高显著,游离胆色素与结合胆色素的比值(t=3.528,P＜0.01)亦明显升高。致石组胆汁中总胆汁酸水平明显降低(t=4.972,P＜0.01)。致石组中成石豚鼠与未成石豚鼠相比,胆汁中总胆色素(t=3.307,P＜0.01)及游离胆色素(t=3.447,P＜0.01)亦升高显著,结合胆色素轻度升高,游离胆色素与结合胆色素的比值(t=3.545,P＜0.01)升高明显。成石豚鼠胆汁中总胆汁酸含量(t=8.040,P＜0.01)明显下降。四、液质联用胆汁代谢组学研究结果成石豚鼠与未成石豚鼠胆汁相比,M/Z的质量数为483.2 amu(牛璜石胆酸)、515.8 amu(牛璜胆酸)、484.2 amu、312.7 amu(硬脂酸)的物质含量明显升高,M/Z质量数为277.9 amu(亚麻酸)、295.0 amu、297.1 amu、506.2 amu、449.0 amu(甘氨胆酸)的物质含量明显降低。结论1、饮食诱导的豚鼠胆囊胆色素结石形成过程中,存在肝细胞胆小管侧膜转运蛋白表达改变。2、饮食诱导的豚鼠胆囊胆色素结石形成过程中,出现胆汁成分的改变,表现为胆汁中游离胆色素含量及比值、牛璜胆酸、牛璜石胆酸、硬脂酸等的含量增高,总胆汁酸、甘氨胆酸及亚麻酸等的含量下降。3、胆色素结石的形成与肝细胞胆小管侧膜转运蛋白异常表达具有一定相关性;肝细胞胆小管侧膜转运功能异常导致胆汁成分变化可能是肝细胞原发性分泌功能参与影响胆色素结石形成的机制之一。4、液质联用进行胆汁代谢组学研究可行。更多还原

【Abstract】 The mechanism of bile pigment stone formation has always been an hot topic in the field of disease of biliary tract over the world.It was reported that changes of the components of bile and bile duct infection played a central role in the bile pigment stone formation.Previous studies were always based on the Maki theory that bile infection was the center mechanism,and analyzed of the composition of bile,gallstone. It was often neglected that infection can lead to the changes of primary liver cells secretion function and original bile components.Recently,scholars carried out extensive research about pigment stones pathogenesis related to immunization,trace elements,oxygen free radicals,amino acids,free fatty acid and content of metal elements.However no breakthrough was found.But research shows that there are three necessary conditions and a central link with the formation of pigment stones.The necessary condition for pigment stones is free bile pigment and calcium increased in bile while the melting factor weakened.The unbalance between the sediment and dissolved bilirubin calcium is the central link of pigment stone.The main components of bile are cholesterol,bile acids and phospholipids,they are all excreted from the liver cell canalicular membrane into the bile.Bilirubin is transported by the liver cells into bile through the same method.Little is known about changes in the composition of bile owing to the primary state change of liver cells secretion in the process of pigment stone formation.We have observed the changes of major components in bile in the pigment stone formation process through the establishment of guinea pig model of pigment gallstone.We also have studied the changes in the expression of liver cell membrane canalicular transporter occurred in the pigment stones,aimed at further exploration about pigment stone formation mechanism from the state of primary liver secretion.In addition bile metabolomics was studied with liquid chromatographymass spectrometry.Through the comparative study of bile in guinea pigs with and failed stone formation,biliary metabolites had been clearly separated in different groups.A further analysis of metabolites contributed to the separation was performed, the initial identification of metabolites with larger difference was produced in pigment stones formation,to speculate the possible molecular formula and structure,and to explore the role of them in pigment gallstone formation.Materials and methodsAnimal modelHealthy 45 guinea pigs(weighing 200～250g) were divided randomly into two groups:normal group(CON),pigment gallstone group(PS).15 guinea pigs in CON group,and 30 in pigment gallstone group.6 to 10 guineas were in each cages,daily food was given for 4 to 6 times,and unrestricted to drinking water.Living environment was with room temperature at 20～25℃,relative humidity of 60 to 70%.After feeding adaptation for a week,normal forage and pigment gallstone-forming forage were given to each group.The period of stone permeation is eight weeks.Specimen collectionThe liver tissue,bile and gallstones were collected for detection and stored at -80℃after eight weeks.Targets detection1.The gallstone-forming rate was assessed and gallstone was determined by infra-red spectrum(IR spectrum).When the gallbladder in guinea pigs was anatomized, it can be clearly distinguished with gallstone formation by the naked eye.The qualitative composition of the calculus was determined by IR spectrum potassium bromide-disk technique,and the rate of stone formation was counted.2.Detecting the amount of total bilirubin,direct bilirubin,undirect bilirubin and total bile acid in different group with biochemical methods.3.Observing the distribution of liver cell membrane canalicular transporter with immunohistochemical method,concerning Bile Salt Export Protein(BSEP,Abcb11), divalent bile salt and bile pigment transporter protein(Multidrug resistance-associated protein,Mrp2,Abcc2),phospholipid transfer protein(Multidrug resistance protein, Mdr,Abcb4).4.Expression of liver cell membrane canalicular transporter(Abcb11,Abcc2, Abcb4) were detected with Western-Blot.Membrane protein was extracted from liver tissue specimens,and tested with SDS gel electrophoresis and ECL chemiluminescence method.5.Expression of liver cell membrane canalicular transporter mRNA(Abcb11, Abcc2,Abcg5,Abcg8) were detected with RT-PCR.RNA was extracted from liver tissue specimens.Target gene was amplified.6.Bile metabolomics was detected by LC-MS method.After the application of high-performance liquid chromatographic to separate the bile,and metabolites was softly ionizated,the mass spectrometry analysis was performed.Statistical analysisSPSS11.5 statistical package was adopted for analysis.The statistical data of our research were all in normal distribution.The results were expressed by mean±SD. Pigment gallstone group and control group were compared and two independent samples T-test was conducted.Comparison of the rate was performed by using chi-square exact test,P＜0.05 was refered as significant difference.Result1.Establishment of guinea pig model of pigment gallstoneModel-building situation on the bile pigment stones in the guinea pig,In guinea, the gallstone can be identified easily during operation and the categories of the gallstones were confirmed by infrared spectrometry.Stone-formation rate in PS group(68.42%)was higher compared with that in control group(14.29%) after feeded for 8 weeks.Stone was identified as a pigment stones.2.Changes in the expression of liver cell membrane canalicular transporter(1) Immunohistochemistry results showed that brown paricles were distributed in the liver cells ductular membrane,suggested the expression of ductular hepatocyte lateral membrane protein.Compared with the CON group,the expressions of Abcb11 (t=5.589,P＜0.01) and Abcc2(t=5.468,P＜0.01) were decreased in PS group and shading less;the coloration of Abcb4 increased slightly,but no statistical significance. In PS group,the coloration of Abcb11(t=8.620,P＜0.01) and Abcc2(t=8.620,P＜0.01) were decreased significantly in the guinea pigs with stone fomation compared with the guinea pigs failed stone formation.(2) RT-PCR results showed that the mRNA expressions of Abcb11(t=6.885, P＜0.01) and Abcc2(t=6.794,P＜0.01) were decreased in PS group compared with the CON group;In PS group,the mRNA expressions of Abcb11(t=2.824,P＜0.05) were also decreased significantly in the guinea pigs with stone fomation compared with the guinea pigs failed stone formation.The mRNA expression decreased concerning with Abcc2(t=2.503,P＜0.05) and with statistical significance.The expressions of Abcg5 and Abcg8 were no statistical in mRNA level.(3)Western Blot results showed that the expressions of Abcb11(t=8.412,P＜0.01, P＜0.01) and Abcc2(t=7.894,P＜0.01) were decreased in PS group compared with the CON group;In PS group,the expressions of Abcb11(t=6.042,P＜0.01) and Abcc2 (t=9.764,P＜0.01) were also decreased significantly in the guinea pigs with stone fomation compared with the guinea pigs failed stone formation.The expression of Abcb4 was no difference in all groups.3.Changes of major components in bileThe results of bile with biochemistry test showed that compared with the control group(CON),the guinea pigs in pigment stone group(PS) was proved with the elevation about total bilirubin(t=3.722,P＜0.01) and free bilirubin(t=3.727,P＜0.01) significant.The ratio between free bilirubin and direct bilirubin(t=3.528,P＜0.01) was also increased significantly.The total bile acid was significantly decreased in PS group(t=4.972,P＜0.01).In PS group,the total bile bilirubin(t=3.307,P＜0.01) and free bilirubin(t=3.447,P＜0.01) were also increased significantly in the guinea pigs with stone fomation compared with the guinea pigs failed stone formation. The direct bilirubin was mildly elevated.The ratio between free bilirubin and direct bilirubin(t=3.545,P＜0.01) was also increased significantly.The total bile acid was significantly decreased in stone formation guinea pigs(t=8.040,P＜0.01).4.LC-MS detection of bile metabolomics researchCompared with failed stone formation guinea pigs,metabolites with M/Z for the quality of 483.2 amu,515.8 amu,484.2 amu,312.7 amu was significantly increased, M/Z mass number for 277.9 amu,295.0 amu,297.1 amu,506.2 amu,449.0 amu was decreased significantly.Conclusions1.The expression of liver cell membrane canalicular transporter was changed during the process of diet-induced guinea pig gallbladder bile pigment stone formation; There was a certain relevance betweenpigment gallstone formation and the abnormal expression of liver cell membrane canalicular transporter.2.During the process of diet-induced pigment gallstone formation in guinea pigs, the changes of bile composition occurred.The levels of free bilirubin,taurocholate, stearic acid were significantly increased,and total bile acid,glycocholic acid and linolenic acid are decreased.This played a role in pigment gallstone formation.3.Secretory function of liver cells was involved in the impact on pigment gallstone formation.The abnormal expression of liver cell membrane canalicular transporter lead to the changes in bile composition may be one of the mechanisms of stone formation as the secretion function of primary liver cells involved in pigment gallstone formation.4.It was feasible for bile metabolomics study with LC-MS.更多还原